Archives of Insect Biochemistry and Physiology 22:113-I 32 (1993) Peptidergic Innervation and Endocrine Cells of Insect Midgut DuSan Zitnan, Ivo Sauman, and FrantiSek Sehnal lnstitute of Ecobiology, Slovak Academy of Sciences, Bratislava (D.Z.), and lnstitute of EntomoloRy, Czechoslovak Academy of Sciences, CesE Budtjovicc O.S., F.S.), Czechoslovakia Antibody against FMRFamide reacts with the stomatogastric innervation and with the midgut endocrine cells in the representatives of most insect orders. The innervation was not revealed in Homoptera, Heteroptera, and Hymenoptera, and the endocrine cells were not recognized i n aphids. Other insects exhibited FMRF-amidepositive endocrine cells of both open and closed types. The cells are mostly single, rarely grouped, and are distributed unequally in different midgut regions; some of the cells project cytoplasmic extensions indicative of a paracrine function. Investigations on Galleria revealed that the gut innervation persists during midgut reconstruction in the course of metamorphosis. The endocrine cells are sloughed off into the new gut lumen, but there they maintain their antigenic properties until a new population of endocrine cells becomes detectable. Antisera to most mammalian gastroenteropancreatic peptides react specifically with the innervation and/or the endocrine cells of insect midgut; only antisera to bombesin, neurotensin, secretin, motilin, and insulin failed to react. All insects seem to contain antigens that can be detected with antisera to pancreatic polypeptide, FMRFamide, enkephalins, and vasopressins. Stomatogastric innervation and the endocrine cells of some lepidopterans also possess allatotropinand diuretic hormone-like antigens; stomatogastric ganglia, in particular, a prothoracicotropic hormone-like antigen. 01993WiIey-~iss,Inc. Key words: neuropeptides, gut hormones, stomatogastric nervous system, prothoracicotropic hormone, allatotropin, diuretic hormone Acknowledgments: We express gratitude for the gifts of antisera (Table 2) to Sandoz Crop Protection (Palo Alto, CA), Dr. H. lshizaki (Nagoya University, Nagoya, Japan), Dr. R. Metz (Leuven University, Leuven, Belgium), Dr. V. HoiejSi (Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague), Dr. M. Nishimura (Shinogi Co., Tokyo, Japan), Dr. N. Yanaihara (Shizuda College, Shizuda, Japan),Dr. H. Vaudry (Rouen University, Rouen, France), and Dr. C.J.P. Grimmelikhuijzen (Hamburg University, Hamburg, Germany). Critical reading of the manuscript by Dr. N.A. Granger of the Universityof North Carolina, Chapel Hill is much appreciated. Received December 16,1991; accepted June20,1992. Address reprint requests to FrantiSek Sehnal, Entomologicallnstitute CSAV, BraniSovska31,370 05 CeskC Bud&jovice,Czechoslovakia. 0 1993 Wiley-Liss, Inc. 114 Zittian et al. INTRODUCTION The current boom of research on insect peptidic hormones largely concerns the neurohormones produced in the central nervous system (CNS) and adjacent corpora cardiaca. Other sources of peptidic hormones include the autonomous nervous system and the midgut, as indicated by the finding of peptidergic neurons in the stomatogastric ganglia [1,2] and of cells with peptidergic granules in the wall of the midgut [3-51. The stomatogastric nervous system is diversified within the class of Insecta [ 6 ] ,and it is not clear whether the occurrence of peptidergic neurons in this system is a general phenomenon. The midgut endocrine cells have been examined in a relatively small number of insects and found to contain, similar to the vertebrates, two types of cells. The apical end of the so-called open type endocrine cells extends into the gut lumen, whereas cells of the closed type do not have direct contact with the lumen . The primary aim of our study is (1) to verify whether visceral innervation and both types of endocrine cells occur in all major insect taxa, and (2) to determine whether they persist when larval midgut degenerates and a new midgut is formed in the course of metamorphosis, Numerous immunohistochemical studies support the notion that the peptides of insect stomatogastric nervous system and the midgut endocrine cells are similar to the ”brain-gut hormones” of the vertebrates [8,9]. In the present study we review and complement available data and compare insects with mammals in respect to the localization of comparable peptides in the visceral nerves vs. the endocrine cells. In addition, since most of the gastroenteropancreatic peptidic hormones also occur in the CNS , the digestive tract of insects has been examined for neurohormones hitherto believed to be found only in the insect brain. The neurohormones in question include silkworm bombyxin and PTTH* [ l O , l l ] and tobacco hornworm ATH and DH [12,13]. MATERIALS AND METHODS Insects and Tissue Preparation Insect species representing different orders were either collected in the field or came from laboratory colonies (Table 1). Digestive tracts of adults were examined in Apterygota and Polyneoptera, and of both larvae and adults in the remaining insects. At least five specimens were used in each species. The fate of the gut innervation and of the endocrine cells during metamorphosis was investigated in the waxmoth, Galleria mellonella, in which the gut is totally reconstructed between larval and adult stages [14,15]. Gut was examined in 6-12 h intervals between the start of cocoon spinning (day 6 of the last larval instar) and the pupal ecdysis (day 7.5)’ in 24 h intervals throughout the pupal instar (total length 7 days), and in freshly ecdysed adults. Insects selected as midgut donors were immobilized either by carbon dioxide treatment, by submersion in water, and/or by exposure to 4°C. The midgut was dissected in PBS (0.16 M NaCl with 0.02 M phosphate buffer, pH *Abbreviations used: ATH = allatotropic hormone; BrdU = bromdeoxyuridine; CRF = corticotropin releasing factor; DH = diuretic hormones; HRP = horseradish peroxidase; PBS = phosphatebuffered saline; PP = pancreatic polypeptide; PTTH = prothoracicotropic hormone. insect Midgut Endocrines 1 15 TABLE 1. Taxonomic Affiliation and Sources of Used Insect Species Apterygota Archaeognatha: Lepismachilis notataa Zygentoma: Thermobiu domesticad and Lepisma saccharin& Palaeoptera Ephemeroptera: Cleon ~ p . ~ Odonata: Agrion s p . a (Zygoptera); Sympetrum sp." (Anisoptera) Polyneoptera Blattodea: Nuuphoeta cinered and Blabera craniiferd Mantodea: Mantis religiosaa Orthoptera-Ensifera: Stenopelmatus uscu6 (Gryllacridoidea);Scudderiujurcutub (Tettigonioidea);Gryllus bimaculatus (Grylloidea) Orthoptera-Caelifera: Melanopus biuita tusb and Dissosteira carolinub(Acridoidea) Phasmatodea: Carausius morosusd Embioptera: Hoembia ~ p . ~ Paraneoptera Homoptera: Tibicen canicularia (Cicadoidea);Aphis sp. '(Afhidoidea) Heteroptera: Pyrrhocoris apterusdand Triatoma sanguisuga Oligoneoptera Neuroptera: Chrysopa sp. ';Asculuphus sp.' Coleoptera: Curubus sp." (Adephaga); Tenebrio molitord (Polyphaga) Diptera: Tipula sp." (Nematocera).Dosophila melanogastetl' and Calliphora vicinad(Cyclorrhapha) Lepidoptera: Galleria rnellonellad; Bombyx morid; Lymantria dispar"; Manduca sextad Hymenoptera: Bomhus s p .' (Aculeata) f "Collected around Bratislava, Czechoslovakia;bcollectedaround Burlington, Vermont; 'collected in Irvine, California; %om laboratory cultures. 7.6), rapidly transferred into a fixative, and left overnight at 4°C. Buffered 4% or 8%formaldehyde, and occasionallyBouin solution (forvery soft guts), were used for the whole mounts. The guts designated for sectioning were fixed in Bouin solutions, dehydrated through an ethanol series and chloroform, embedded in paraplast, and cut at 7-10 pm. Immunohistochemistry Normal goat and horse sera were purchased from Dakopatts (Glostrup, Denmark) and Vector Laboratories (Burlingame, CA), respectively. Primary antisera and their suppliers are listed in Table 2. All sera were diluted with PBS containing 0.05% sodium azide and 0.1% Triton X-100; this solution was also used for washing fixed tissues and their sections. Biotin-streptavidin-HRP immunostaining kit (Amersham, Little Chalfont, UK) or the ABC Vectastain kit (Vector Laboratories) were used routinely to reveal primary antisera. Activity of most primary antisera against the vertebrate-type peptides was checked in sections of murine gastroenteropancreatic system. The specificity of antisera against PTTH, bombyxin, ATH, and DH was verified by immunostaining the nervous system of Galleria mellonella and Manduca sexfa, and by using antisera saturated with respective antigens. Deparaffinnized and hydrated sections were treated with 0.1 % hydrogen peroxide in PBS to block endogenous peroxidase activity and preincubated in 5%normal goat or horse serum for 15min to prevent nonspecific IgG binding. Unless stated otherwise, the sections were then incubated at room temperature in the following sequence: ( 1)primary antibody overnight at P C , (2) PBS Twes of antibodies: Gp = Guinea pig; R = rabbit; M = R(143II) R R R R R R( 117III) R(1611) R R R M M M(IN-03) Gp(0002) R R R R Gp(1305) R R Type 1:1,000 1:1,000 1:1,000 1:1,000 1:330 1:500 1:500 1:1,000 1:500 1:500 1:500 1:500 1:500 1:500 1:1,000 1:1,000 1:1,000 1:1,000 1:1,000 1:1,000 1:1,000 1:1,000 Dilution Supplier Sandoz Crop Protection Sandoz Crop Protection Dr. H. Ishizaki Dr. H. Ishizaki Dr. V. HoiejSi Dr. M. Nishimura Dr. R. Metz INCSTAR Dr. N. Yanaihara Dr. N. Yanaihara Dr. N. Yanaihara INCSTAR INCSTAR INCSTAR Dr. R. Metz Dr. H. Vaudry Dr. H. Vaudry Dr. H. Vaudry Dr. H. Vaudry Dr. C.J.P. Grimmelikhuijzen Dr.C.J.P. Grimmelikhuijzen Dr. C.J.P.Grimmelikhuijzen mouse, monoclonal; lot identifications are given in parentheses. Manduca allatotropin Manduca diuretic hormone Bombyx PTTH (N-terminal pentadecapeptide),0.5 mg/ml Bombyx bombyxin I1 (N-terminal decapeptide), 0.5 mg/ml Insulin, 0.1 mg/ml Insulin Glucagon Vasoactive intestinal peptide (VIP) Peptide histidine isoleucine (PHI) Motilin Gastrin Cholecystokinin 8 Bombesin Somatostatin Neurotensin a-Melanostimulating hormone (aMSH) @-Endorphin Met-enkep halin Leu-enkephalin FMRFamide Bovine pancreatic polypeptide (C-terminal hexapeptide amide) Arginine vasopressin Antigen TABLE 2. List of Prirnarv Antibodies* Insect Midgut Endocrines 117 wash, (3) secondary biotin-coupled antibody (diluted 1:200), 1 h, (4)PBS wash, (5) biotin-streptavidin-HRP (1:200) or avidin-HRP (1:200), 1 h, (6) washing in 0.1 M Tris-HC1, pH 7.6. With the guinea pig primary antisera, goat antiguinea pig IgG conjugated with HRP (Nordic, Tilburg, The Netherlands) was used as secondary antibody (step 3), followed by washing in Tris-HC1buffer. Peroxidase activity was revealed by incubating the sections in 0.01% diaminobenzidine (Sigma, St. Louis, MO) and 0.0003% hydrogen peroxide in 50 mM Tris-HC1buffer, pH 7.6. The sections were then slightly counterstained with diluted Mayer's hernatoxylin for 1-2 min, washed in distilled water, and mounted in glycerin-gelatin (30% and 7% in water). For the whole mounts, the fixed guts were cut laterally and cleansed of contents, thoroughly washed in PBS, incubated for 1-2 h in 5% normal goat or horse serum, and immunostained in the following sequence: (1)primary antiserum against FMRFamide for 48 h at 4"C with occasional shaking, (2)four or more PBS washes, 15 min each, (3) secondary biotinylated antiserum (1:200), overnight at 4"C, (4)four washes in PBS, 15 min each, ( 5 )avidin-HRP (1:200) or biotin-streptavidin-HRP (1:ZOO) for 4-6 h, (6) three washes in PBS, and one in 0.1 M Tris-HC1 (pH 7.6), 15 min each. Peroxidase activity was detected as described above. Guts were washed in PBS-glycerol (l:l),spread on a slide, and mounted in glycerin-gelatin. BromdeoxyuridineLabelling and Double Immunohistochemical Staining BrdU labelling was used to mark cells synthesizing DNA in preparation for the cell division. Postfeeding waxmoth larvae were water-anaesthetized and injected (0.5 pl per 50 mg body weight) with 0.33% BrdU (Sigma) in EphrussiBeadle s a h e (prepared freshly from a stock solution of 3.3% BrdU in 30% ethanol).The midgut was dissected after 2 h, fixed in Bouin's solution, embedded in paraplast, and cut at 7 pm. Rehydrated sections were pretreated for 30 min with 2 N HCl in PBS to denature DNA, and after treatmentswith 0.1% hydrogen peroxide and 10% goat serum, they were double-immunostained according to the followingprotocol: (1)a mixture of monoclonal anti-BrdU IgG antibody (1:30), and the rabbit anti-FMRFamide antiserum (1:1,000), overnight at 4"C, (2) three washes in PBS, 2 min each, (3) a mixture of goat antimouse IgG conjugated to HRP (1:200), and goat antirabbit IgG conjugated to alkaline phosphatase (1:200), 1h (antibodies from Vector Laboratories), (4)two washes in PBS, one wash in 0.1 M Tris-HC1 (pH 7.6), 2 min each, (5) detection of peroxidase activity as described above, (6) wash in 0.1 M Tris-HC1, pH 8.5, 5 min, (7) detection of alkaline phosphatase activity with a mixture containing 15mg naphthol-AS-MXphosphate in 1rnl N,N-dimethylformamide, 50 mg fast blue BB salt, and 24 mg Levamisole in 70 ml 0.1 M TRIS-HC1buffer, pH 8.5, (8) washing in water and mounting in glycerol-gelatin. RESULTS Innervation and Endocrine Cells in the Midgut of Different Insects The antiserum against FMRFamide was used to compare the morphology of midgut innervation and the occurrence of midgut endocrine cells in species 118 Zitnan et al. listed in Table 1. In the following description, the results are related to insect subclasses and orders, to which the examined species belong. The data on innervation concern only that part of the stomatogastric nervous system which is posterior to the oesophageal nerve. In Apterygota, either a nonbranching (Archaeognatha) or branching (Zygentoma) oesophageal nerve runs along the dorsal foregut surface to a single ingluvial ganglion at the foregut/midgut junction. The ganglion contains 4-15 immunopositive cells and sends off either one dorsal and one ventral (Archaeognatha), or several (Zygentoma) nerves that arborize over the midgut surface into a plexus. Characteristic features of the midgut endocrine cells include their exclusive localization in the center or close to the center of regenerative nidi and their occasional arrangement in groups of 2-5. The morphology and the grouping of cells vary in differentzones of the midgut. For example, in Lepismachilis, the foremost part of the midgut is devoid of cells reactive with FMRFamide antibody, whereas a zone comprising about fourfifths of the gut length contains closed-type cells of amoeboid shape; the last fifth of the midgut accommodates mostly open-type cells, along with small, round cells of the closed type. In our study of Palaeoptera, the representatives of mayflies (Ephemeroptera) exhibited very weak immunoreactivity that was detectable only in the varicosities of gastric nerves. Strongly reacting endocrine cells were seen at the very beginning of the midgut (mostly closed-type cells) and in the middle third of its length (mostly long cells of the open type), whereas other midgut regions seemed devoid of the FMRFarnide-positive endocrine cells. In contrast, the antibody against FMRFamide revealed a distinct midgut innervation and a widespread occurrence of the endocrine cells in the damseflies and dragonflies (Odonata). A branching oesophageal nerve terminates in the ingluvial ganglion that is connected to a pair of proventricular ganglia, which supply the midgut with a meshwork of anastomosing nerves. The endocrine cells are singly scattered and of both the open and closed types; the closed cells often possess cytoplasmic projections and are presumably paracrine. The density of endocrine cells is highest in the central and in the posterior portions of the midgut. Immunoreactivity to FMRFamide antibody was very good in all representatives of Polyneoptera. In the cockroaches (Blattodea), the oesophageal nerve, which lacks immunoreactive neurons, splits at the frontal crop region into a dorsal and a ventral ingluvial nerve. The branching point, which was regarded as a rudimental ingluvial ganglion , consists of 1 4 immunoreactive perikarya that are also persent in large numbers along the ingluvial nerves (Fig. 1A). These send fine branches over the crop and at its posterior margin they ramify into a number of gastric nerves, providing the meshwork innervation of the midgut (Fig. 1B). The endocrine cells are distributed singly throughout the midgut. In contrast to some other insects, they lack the cytoplasmic processes. In the praying mantis (Mantodea), the walking stick (Phasmodea), and the embiens (Embioptera), a single dorsal oesophageal nerve with fine side branches terminates in a distinct ingluvial ganglion containing immunoreactive perikarya. In the walking stick, the perikarya also occur in the vicinity of Insect Midgut Endocrines 1 19 Fig. 1. Gut innervation and midgut endocrine cells revealed with FMRFamide antibody in Polyneoptera and Paraneoptera. A: Perikarya (arrows) in the oesophageal nerve (ON) and the ingluvial nerves (IN)of Nauphoeta. B: Anastomosinggastric nerves on the anterior region of midgut in Blabera. C: Proventricular ganglion (PG) in Hoernbia. D: A neuron (N) on midgut surface, and a closed endocrine cell (EC) with fine paracrine processes in Carausius. E: Clusters ( C ) of closed endocrine cells in midgut nidi, and apparently holocrine immunoreactive secretion (arrows) in the gut lumen of Gryllus. F: Dominating open-type endocrine cells with a long apical extension in the anterior region of midgut in Dysdercus. A-D, F, whole mounts, E, section. Bars = 50 prn. the ingluvial ganglion on the foregut surface, as well as along the gastric nerves in the proximal midgut region (Fig. 1D). Gastric nerves emanate from the ingluvial ganglion in the praying mantis and the walking stick, whereas embiens possess a pair of proventricular ganglia; the gastric nerves start there 120 iitnan et aI. (Fig. IC). A plexus of anastomosing gastric nerves on the midgut surface is very obvious. At least some of the midgut endocrine cells are of the closed type and have an amoeboid shape with cytoplasmic projections; such cells occur only in the caeca in the praying mantis but in different midgut regions in Phasmodea and Embioptera. In the walking stick, the endocrine cells of both closed and open types are of asteroid shape and their paracrine projections point in all directions (Fig. 1D). Although the females of Embioptera contain both closed and open endocrine cells, only the closed cells are found in the males. Paired oesophageal nerves are a common feature of Orthoptera. The two branches of the nerve begin laterally in the hypocerebral ganglion and run in a spiral fashion around the crop to a dorsal and a ventral ingluvial ganglion. In the suborder Ensifera, each of these ganglia contains about 15immunopositive cells and sends off one ingluvial nerve supplying the crop and one caecal nerve that arborizes over the surface of the two caeca. The caecal nerve splits into single or paired dorsal and ventral gastric nerves. By contrast, each ingluvial ganglion of locusts (suborder Caelifera), includes only 2-3 immunopositive cells, whereas additional cells lay along several ingluvial and two caecal nerves that extend from each ganglion. The caecal nerves split into numerous gastric nerves that anastomose over the midgut surface. The distribution and shape of the endocrine cells of both open and closed types vary along the midgut length. Singly scattered, round cells of the closed type are most common. In the cricket, however, a midgut zone just behind the caeca contains also clustered endocrine cells (Fig. 1E). The majority of the endocrine cells in the caecal region of locusts are not round, but possess cytoplasmic extensions. Paraneoptera seem to differ from other insects by the lack of immunoreactivity to FMRFamide antiserum in midgut innervation, indicating that the gastric nerves of Homoptera and Heteroptera are either reduced or devoid of FMRFamide-like peptides; a similar observation was made on the bug Rhodnius prolixus . Both open and closed endocrine cells can be visualized with the FMRFamide antiserum in the cicada and bugs, but no endocrine cells were discerned in the aphids. In the cicada, some of the cells are paired; some of the single cells appear paracrine. A characteristic feature of the bugs are very long, open-type cells in the anterior sack-like region of the midgut (Fig. 1F). Gut innervation of some Oligoneoptera also reacts poorly with the FMRFamide antibody. In Neuroptera, there are two oesophageal nerves that send branches over the crop, and at the end of the crop they dichotomize into gastric nerves. Only varicosities of these nerves show clear immunoreactivity in Chrysopa. About 10 perikarya occur at the rear of the foregut in Crysopa, whereas up to 20 neurons are localized along the gastric nerves in the posterior quarter of the midgut in Ascalaphus. Endocrine cells are mostly rounded, but in the posterior half of the midgut in Ascaluphus they are occasionally amoeboid and possess long cytoplasmic processes linking them to one another and to the innervation. In Chrysopa, the closed-type cells are occasionally paired. In the Coleoptera, Curubus showed no immunoreactivity in the foregut region and only weak reaction in four gastric nerves. In Terzebrio, the innervation was clearly stained: a single oesophageal nerve arborizes at the end of Insect Midgut Endocrines 121 the foregut into a meshwork containing about 20 perikarya and a number of gastric nerves run from there over the midgut length. Endocrine cells are both open and closed types, and some of the latter possess cytoplasmic extensions. In the examined Diptera, a pair of oesophageal nerves terminate in the two ingluvial ganglia, each of which contains 4 8 immunoreactive perikarya. The ganglia supply a total of 4 4 gastric nerves that can be traced along the entire midgut length in Tipula, but only in gastric caeca in Drusophila . Endocrine cells are large, rounded, and, in Drosophifa, mostly of the open type. A common feature of Lepidoptera seems to be the single oesophageal nerve terminating in the ingluvial ganglion, which innervates either a pair of proventricular ganglia (Galleria) or a loose conglomeration of neurons in the first quarter of the midgut (Bombyx, Lymantria, Munduca; see Fig. 5F). Eight gastric nerves begin in the foregut/midgut junction in most species, but in Lyman tria, numerous gastric nerves appear to extend from individual neurons located in the anterior region of the midgut (see Fig. 5E). Open bottle-shape and closed rounded endocrine cells are singly scattered throughout the midgut epithelium, with increasing density toward the end of midgut. In the only representative of Hymenoptera, the nerves were not stained, but about 20 neurons were revealed along the longitudinal muscles in the foremost part of the midgut. Rounded endocrine cells, mostly of the open type, occur singly throughout the midgut. Reconstruction of Gut Endocrines During Metamorphosis Gut innervation and midgut endocrine cells are found in the larvae, pupae, and adults of oligoneopterans, including those in which the larval digestive tract degenerates during metamorphosis and is replaced by a new one. Using double-immunostaining, we followed the process of midgut replacement and the fate of endocrine cells in Galleria. The dividing regenerative cells were marked with BrdU, whereas the endocrine cells were recognized with the anti-FMRFamide antiserum. No incorporation of BrdU was detected in the wandering larvae, but it was clear in those that had initiated cocoon spinning. By comparing a series of insects that were injected with BrdU between spinning initiation and the completion of apolysis (pharate pupal stage), we deduced that midgut reconstruction proceeds as portrayed in Figure 2 (to avoid confusion in the black-and-white photographs, preparations treated only with the anti-FMRFamideantiserum are shown). Larval midgut consists of a single layer epithelium containing nidi of regenerative cells, digestive goblet and columnar cells, and sparsely scattered endocrine cells (Fig. 2A). The regenerative cells begin to incorporate BrdU in the middle of the cocoon-spinning period. The BrdU-labelled cells occur throughout the midgut length, and their number rapidly increases. They remain attached to basal lamina while the surrounding midgut epithelium detaches and is pushed into the gut lumen (Fig. 28). The regenerative cells continue to proliferate and in the postspinning stage, the slowly mobile prepupae form the continuous epithelium of the pupal midgut (Fig. 2C). At this stage, the new midgut contains very few FMRFamide-positive cells. The old larval midgut begins to disintegrate; however, some of the FMRFamide-positive cells 122 iitnan et al. Fig. 2. Changes in the endocrine cells (arrows) during midgut metamorphosis in Galleria (sections, antibody to FMRFamide). A: Functional midgut of a feeding larva. 6: Larval gut epithelium (LG) with endocrine cells, enclosed by newly differentiating pupal gut (PG), in a larva at the end of cocoon spinning. C: Degenerating larval gut (LG) and pupal gut (PC) in a pharate pupa. D: Midgut with an imrnunopositive cell 24 h after pupal ecdysis. E: Disintegrating cells of pupal midgut (PG) with remaining regenerative and endocrine cells in a pharate adult; F: Adult midgut. Bars = 50 prn. appear to be preserved in the degenerated larval midgut until after pupal ecdysis. The number of endocrine cells increases during the pupal stage (Fig. 2D), and they remain preserved when the pupal midgut degenerates and the imaginal midgut is formed (Fig. 2E,F). Vertebrate-Type Regulatory Peptides in the Innervation and Endocrine Cells of Insect Midgut Examples of the immunohistochemical detection of mammalian gastroenteropancreatic peptidic hormones in the gut of insects are provided in Figure 3, and the distribution of these peptides in the innervation and the endocrine cells of several insects is compared with mammals in Table 3. Insect Midgut Endocrines 123 In all examined insects, both the gut innervation and the endocrine cells react with antisera against PP, P-endorphin, enkephalins, FMRFarnide, and vasopressin; in addition to species listed in Table 3, we detected this reaction also in the representatives of locusts, crickets, and phasmids (data not shown). These are the only antisera that yielded a positive reaction in bugs and flies we examined, but the tse-tse fly was reported not to respond to the PP Fig. 3. lrnrnunodetection of different regulatory peptides in the endocrine cells of the midgut (sections). A Group of endocrine cells revealed with antiserum against Arg-vasopressin in lepisma. 6: Reactionwith antiserum against Met-enkephalin in the nidi of Lepisma. C: Gastrin-irnmunopositive cells in the anterior region of midgut in Nauphoeta. D: Cholecystokinin-positive cells in the posterior region of midgut in Nauphoeta. E,F The same midgut, reactions with antisera against Arg-vasopressin and peptide histidine isoleucine, respectively. C: Endocrinecell revealed with antiserum against FMRFarnide in pyrrhocoris. H: Endocrine cells containing pancreatic polypeptide-like antigen in Calliphora. Bars = 50 p. + - - ? ? - - - t + + + + + + ? - ? ? ? - + + - + - + ? ? + ? ? Lepisma N E + ? ? + ? ? ? ? + + + + ? ? + ? ? + + ? ? ? ? + + + - ? ? + ? ? Aeschna N E + + + + + + + + + + + + f f + + + + + + + + + + + + + + Roaches N E + + + + - ? ? - ? + - - - ? ? ? ? ? - ? ? ? + + - - + + + + - - - + - + - - ? ? - + + + - ? ? - ? - - + - ? ? - Calliphora N E - - ? ? - Pyrrhocoris N E + + + + ? ? ? - - + + + - ? ? - ? - + + + + - + + + + - - Galleria N E - + + + + + + + + + + + - + + + + + + + + + + + + + Mammals N E *Listed insects: Silverfish Lqisrna saccharins; dragonfly Aeschna cyunea, data [18,19]; cockroaches: A combination of data on Peripluneta arnericanu [20,21], Blaberits cmnirfir  and Nauphoefa cinerea (our results); bug Fyrrhocuris apterus; fleshfly Calliphora vicina; waxmoth Galleria rnellonella. Data on peptide localization in the gut innervation in Pyrrhocoris concern the stomodeal nervous system (gastric nerves were not revealed). Distribution of regulatory peptides in the gut of mammals is taken from [2%25]. Abbreviated peptide names: CKF = corticotmpin releasing factor; ACTH = adrenocorticotropin; MSH = melanization stimulating hormone; + = present; - = absent; ? = not tested; for other abbreviations see list of Abbreviations used. Glincentin Glucagon VIP PHI Gastrins Cholecystokinin PP Somatostatin Substance P Neuro tensin CRF ACTH a-MSH P-Endorphin Enkephalins FMRFamide Vasopressin Peptide TABLE 3. Immunohistochemical Identification of Gastroentoropancreatic Peptides in the Gastric Nerves (N)and Midgut Endocrine Cells (E)of Representative Insects and Occurrence of These Peptides in Mammalian Gut* Insect Midgut Endocrines 125 antiserum . Most insects appear to contain antigens to additional vertebrate-type regulatory peptides. Antiserum against cholecystokinin reacts in the gut of dragonflies, locusts, cockroaches, phasmids, lepidopterans, and beetles (Table 3) . The greatest variety of antigens was found in the gut of cockroaches. Insects do not seem to contain neurotensin-like antigens in the gut (Table 3) and possibly also lack peptides related to bombesin, secretin, and rnotilin (data not shown), which are characteristicfor the gut innervation (bombesin) and the endocrine cells, respectively, of mammalian gut. We also failed to detect any specific insulin-like immunoreactivity (see Discussion). Just as in mammals, some of the regulatory peptides are present both in the innervation and in the endocrine cells of insect gut, whereas others occur only in one of these peptidergic sources. Glicentin- and glucagon-like peptides seem to be confined to the endocrine cells in both mammals and insects, but gastrins, CRF, and substance P, which are also specific for certain endocrine cells in mammals, are immunohistochemically detectable both in the endocrine cells and in the gut innervation in insects (Table 3). Neuropeptide Y, which is present in mammals exclusively in neurons, was identified both in the nerves and in the endocrine cells of Locusta migratoria . Finally, the peptide His-Ileu appears in the gut innervation of mammals, whereas in insects it was immunohistochemically detected in the endocrine cells (Table 3). The following regulatory peptides, which are believed to be restricted in vertebrates to the CNS, were immunohistochemicallydetected in both nerves and endocrine cells of the insect gut: vasopressin-like antigens were revealed in all examined species (Table3), and antibody to urotensin I reacted in Gryllus and Periplaneta . Even more surprising, the hypothalamic growth hormone-releasing factor and the luteinizing hormone-releasing factor occur in the dragonfly A . cyanea and in the cockroach B . craniifer in the endocrine cells but not in the nerves of the gut [19,21]. Insect Neurohormones in the Innervation and in the Endocrine Cells of the Midgut We never found a reaction with bornbyxin antibody, which identified specific neurons in the CNS [10,30], in the digestive tracts of several lepidopterans. The antibody against PTTH reacted with certain neurons in the stomodeal nervous system and with gastric nerves, but not with the endocrine cells (Table 4). Antisera against ATH and DH, however, recognized antigens both in the innervation and in the endocrine cells of Galleria, Manduca (Table 4), and Lymantria (data not shown). Antisera against PTTH and ATH displayed identical pattern of staining in the neurons and nerves (Fig. 4A,B). A more detailed study was performed with last instar larvae of Manduca, the species from which ATH and DH had been isolated [12,13]. Both the innervation (Fig. 4C,D) and the endocrine cells (Fig. 4G,H) react with these antisera. DH-like antigen occurs in a higher number of ganglionic neurons than the ATH-like antigen, but it is wanting in the enteric plexus, in which ATH-like antigen appears to be present in large amounts (Fig. 4F). A similar distribution of ATH-immunoreactivity was found in Lymantria (Fig. 4E). In spite of the lack of DH-like antigen in the enteric plexus, where the gastric 126 Zitnan et al. TABLE 4. Occurrence of Insect Neuropeptides in Gut Innervation and Midgut Endocrine Cells Species and peptides G. mellonella P'ITH ATH DH M.sextu PTTH ATH DH Localizationa Frontal ganglion 2 2 2 Frontal ganglion 2 24 3+4 aNumbers of immunoreactive perikarya; Ingluvial ganglion 24 2 4 ? Hypocerebral ganglion - 4.4 Proventricular ganglion 4-6 4 ? Endocrine cells - + ? Enteric plexus Endocrine cells + + - - + + = presence, - = absence of reactivity in the nerves. nerves appear to originate, immunostained axons are found over the entire midgut surface (Fig. 4H). DISCUSSION There is little doubt that peptidergic gut innervation and peptidergic midgut endocrine cells occur in all insects. With a few exceptions, they can be visualized with the antisera against FMRFamide. In our study, only the innervation of foregut in Curubus and Bornbus, the innervation of midgut in Puruneopteru, and the endocrine cells in aphids failed to respond to FMRFamide antiserum. The midgut innervation emanates from the stomodeal nervous system, the arrangement of which is characteristic for insect orders or sub-orders [ 6 ] .The posterior ganglia (ingluvial and proventricular) are often replaced by loose neurons; in Manduca, their assembly is called the enteric plexus . Gastric nerves, which in some species include perikarya, appear to begin in the posterior ganglia or in the enteric plexus , but the distribution of DH-like antigen (Table 4) suggests that they alsocontainaxons from the frontal ganglion or from the CNS. Gastric nerves form a network of fine fibers in midgut musculature, and there is evidence that gastric nerves link the stomodeal nervous system with the proctodeal innervation . Axons supplying midgut muscles were shown to contain a variety of neurosecretory granules . As most other animals, the insects contain endodermal endocrine cells of both open and closed types . The cells are usually distributed singly through most or all of the midgut, but in the apterygotes, crickets and Chrysopu, some of them occur in groups [5, and our observations]. Endocrine cells are derived from the regenerative nidi, which also generate the digestive cells 136, and our data]. Morphology, size, and abundance of the endocrine cells are different in different insect taxa, and in various midgut regions of the same species. Differences in the immunoreactivity of the endocrine cells in Insect Midgut Endocrines 127 Fig. 4. lmrnunodetection of insect neurohormone-like antigens. Allatotropin-like (A) and PTTH-like antigens (B) in the frontal ganglion of Galleria larvae (A, end of the penultimate, i.e., 6th instar; 6 , beginning of the 6th instar). Allatotropin-like (C) and diuretic hormone-like antigens (D) in the frontal ganglion of wandering Manduca larvae; immunoreactive neurons (arrows) are present also in the hypocerebral ganglion (HG). Allatotropin-like antigen in the nervous midgut plexus of fresh Lymantria pupa (E) and of wandering Manduca larva (F); arrows point to perikarya. Allatotropin-like (C) and diuretic hormone-like antigens (H)in the fine nerves ( N ) and in the endocrine cells (arrow) of the feeding (C) and wandering (H) Manduca larvae. Bars 50 pm. 128 Zitnan et al. various insect groups are documented in Table 3. Ultrastructural [22,34,37,38], immunohistochemical [20,28], and immunocytochemical  evidence is available for the diversity of the endocrine cells within the midgut of a single species. Widespread occurrence, chemical diversity, and structural complexity, which are maintained when the gut is rebuilt at metamorphosis, indicate that the innervation and the endocrine cells of the gut are of great importance. The information on mammals demonstrates that the two systems are interlinked and jointly communicate with the CNS . According to Fujita et al. , the endocrine cells function as primary sensors, those of the open type registering the nutrient contents of the gut, and those of the closed type perceiving the tension in the gut wall. Upon appropriate sensory stimulation, the endocrine cells release their secretions primarily into their immediate vicinity (paracrine secretion). The secretions act mostly locally, affecting gut movements, production of digestive fluids, rate of replacement of the gut epithelium, and blood flow to the gut. There is some evidence that the secretion is released into the gut lumen and acts on the apical sites of the digestive cells. Paracrine secretions apparently act also on the nerve termini in the subepithelial nervous reticulum, by which the humoral signal is transduced into nervous stimuli and eventually causes changes in the nervously controlled functions, including behavior. Finally, some products of the endocrine gut cells enter the body circulation and exert hormonal effects on distal targets. The increasing knowledge of insect peptidic hormones is consistent with the idea that most of them belong to the same peptide families as the hormones of vertebrates . Even though the immunohistochemical data cannot provide a proof of chemical identity and may lead to false conclusions , the consistent demonstration of a number of vertebrate peptides in various insects and with different antibodies and techniques suggests that most of the peptidic hormones in insect gut innervation and midgut endocrine cells are similar to those of the vertebrate gut. The endocrine system of insect gut parallels that of the vertebrates also in peptide localization in the nerves vs. the endocrine cells, which seems to be reversed only in the case of peptide His-Ileu (Table 3). Antisera to neurotensin, secretin, and motilin, which are the dominant peptides in the gut of mammals, do not react in the insect gut. A reaction with antiserum against bombesin, another common mammalian peptide, was reported in a single study of a locust 1391. Secretion of an insulin-like material in the digestive tract of insects is a subject of dispute. Whereas radioimmunoassays and metabolic bioassays of midgut extracts have indicated the presence of insulin-like protein(s) in several insects [43-46], the immunohistochemical approach has yielded a positive response in one case only 1391. Differences in the content of midgut hormones among the insect taxa (Table 3) resemble the situation in the vertebrates . Even in mammals, the physiological effects of gastroenteropancreatic peptides are far from being fully elucidated, and it is becoming clear that in many cases they are complex. For example, cholecystokinin controls gall bladder contractions, pancreatic enzyme secretion, and gastric emptying, and via the latter effectit inhibits food intake . Multiple roles of gut secretions are also Insect Midgut Endocrines 129 indicated by the data on insects. Involvement in the control of food processing was proposed by Brown et al. , who showed that the number of immunoreactive cells and their content of FRMFamide-like and PP-like material in the midgut of adult mosquito females decreases after the blood meal, sugesting depletion of the regulatory peptides. By contrast, in larval corn earworms, Heliothis zed, feeding evokes an increase in FMRFamide immunoreactivity in the endocrine cells, whereas the blood concentration of the immunoreactive agent is higher in starved insects . 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